Isolated cancer associated antigens NY-MEL 1 and NY-MEL 3, peptides based thereon, and their uses

ABSTRACT

The invention relates to isolated, cancer associated antigens, nucleic acid molecules, and various uses thereof.

FIELD OF THE INVENTION

[0001] This invention relates to antigens associated with cancer, thenucleic acid molecules encoding them, as well as the uses of these.

BACKGROUND AND PRIOR ART

[0002] It is fairly well established that many pathological conditions,such as infections, cancer, autoimmune disorders, etc., arecharacterized by the inappropriate expression of certain molecules.These molecules thus serve as “markers” for a particular pathological orabnormal condition. Apart from their use as diagnostic “targets”, i.e.,materials to be identified to diagnose these abnormal conditions, themolecules serve as reagents which can be used to generate diagnosticand/or therapeutic agents. A by no means limiting example of this is theuse of cancer markers to produce antibodies specific to a particularmarker. Yet another non-limiting example is the use of a peptide whichcomplexes with an MHC molecule, to generate cytolytic T cells againstabnormal cells.

[0003] Preparation of such materials, of course, presupposes a source ofthe reagents used to generate these. Purification from cells is onelaborious, far from sure method of doing so. Another preferred method isthe isolation of nucleic acid molecules which encode a particularmarker, followed by the use of the isolated encoding molecule to expressthe desired molecule.

[0004] Two basic strategies have been employed for the detection of suchantigens, in e.g., human tumors. These will be referred to as thegenetic approach and the biochemical approach. The genetic approach isexemplified by, e.g., dePlaen et al., Proc. Natl. Sci. USA 85: 2275(1988), incorporated by reference. In this approach, several hundredpools of plasmids of a cDNA library obtained from a tumor aretransfected into recipient cells, such as COS cells, or intoantigen-negative variants of tumor cell lines which are tested for theexpression of the specific antigen. The biochemical approach,exemplified by, e.g., O. Mandelboim, et al., Nature 369; 69 (1994)incorporated by reference, is based on acidic elution of peptides whichhave bound to MHC-class I molecules of tumor cells, followed byreversed-phase high performance liquid chromography (HPLC). Antigenicpeptides are identified after they bind to empty MHC-class I moleculesof mutant cell lines, defective in antigen processing, and inducespecific reactions with cytotoxic T-lymphocytes. These reactions includeinduction of CTL proliferation, TNF release, and lysis of target cells,measurable in an MTT assay, or a ⁵¹Cr release assay.

[0005] These two approaches to the molecular definition of antigens havethe following disadvantages: first, they are enormously cumbersome,time-consuming and expensive; and second, they depend on theestablishment of cytotoxic T cell lines (CTLs) with predefinedspecificity.

[0006] The problems inherent to the two known approaches for theidentification and molecular definition of antigens is best demonstratedby the fact that both methods have, so far, succeeded in defining onlyvery few new antigens in human tumors. See, e.g., van der Bruggen etal., Science 254: 1643-1647 (1991); Brichard et al., J. Exp. Med. 178:489-495 (1993); Coulie, et al., J. Exp. Med. 180: 35-42 (1994);Kawakami, et al., Proc. Natl. Acad. Sci. USA 91: 3515-3519 (1994).

[0007] Further, the methodologies described rely on the availability ofestablished, permanent cell lines of the cancer type underconsideration. It is very difficult to establish cell lines from certaincancer types, as is shown by, e.g., Oettgen, et al., Immunol. Allerg.Clin. North. Am. 10: 607-637 (1990). It is also known that someepithelial cell type cancers are poorly susceptible to CTLs in vitro,precluding routine analysis. These problems have stimulated the art todevelop additional methodologies for identifying cancer associatedantigens.

[0008] One key methodology is described by Sahin, et al., Proc. Natl.Acad. Sci. USA 92: 11810-11913 (1995), incorporated by reference. Also,see U.S. Pat. No. 5,698,396, and application Ser. No. 08/479,328, filedon Jun. 7, 1995 and Jan. 3, 1996, respectively. All three of thesereferences are incorporated by reference. To summarize, the methodinvolves the expression of cDNA libraries in a prokaryotic host. (Thelibraries are secured from a tumor sample). The expressed libraries arethen immunoscreened with absorbed and diluted sera, in order to detectthose antigens which elicit high titer humoral responses. Thismethodology is known as the SEREX method (“Serological identification ofantigens by Recombinant Expression Cloning”). The methodology has beenemployed to confirm expression of previously identified tumor associatedantigens, as well as to detect new ones. See the above referenced patentapplications and Sahin, et al., supra, as well as Crew, et al., EMBO J144: 2333-2340 (1995).

[0009] This methodology has been applied to a range of tumor types,including those described by Sahin et al., supra, and Pfreundschuh,supra, as well as to esophageal cancer (Chen et al., Proc. Natl. Acad.Sci. USA 94: 1914-1918 (1997)); lung cancer (Güre et al., Cancer Res.58: 1034-1041 (1998)); colon cancer (Ser. No. 08/948,705 filed Oct. 10,1997) incorporated by reference, and so forth. Among the antigensidentified via SEREX are the SSX2 molecule (Sahin et al., Proc. Natl.Acad. Sci. USA 92: 11810-11813 (1995); Tureci et al., Cancer Res. 56:4766-4772 (1996); NY-ESO-1 Chen, et al., Proc. Natl. Acad. Sci. USA 94:1914-1918 (1997); and SCP1 (Ser. No. 08/892,705 filed Jul. 15, 1997)incorporated by reference. Analysis of SEREX identified antigens hasshown overlap between SEREX defined and CTL defined antigens. MAGE-1,tyrosinase, and NY-ESO-1 have all been shown to be recognized by patientantibodies as well as CTLs, showing that humoral and cell mediatedresponses do act in concert.

[0010] It is clear from this summary that identification of relevantantigens via SEREX is a desirable aim. In U.S. patent application Ser.No. 09/451,739, filed Nov. 30, 1999 and incorporated by reference,various antigens have been identified, and nucleic acid moleculesencoding these have been isolated. The inventors have applied themethodologies described supra and have identified several new antigensassociated with cancer, as detailed in the description which follows.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS EXAMPLE 1

[0011] Cell line Mz19 was used in this example. It was derived from amelanoma patient, from whom the autologous serum referred to infra wasalso taken. Total RNA was extracted from cell samples, using standardCsCl guanidine isothiocyanate gradient methodologies. A cDNA library wasprepared, in λ ZAP, following standard methods. The resulting library of7×10⁵ primary clones was then analyzed using the SEREX methodology of,e.g., Sahin, et al., Proc. Natl. Acad. Sci. USA 92:11810-11813 (1995);Chen, et al., Proc. Natl. Acad. Sci. USA 94: 1914-1918 (1997), andPfreundschuh, U.S. Pat. No.5,698,396. These references are allincorporated by reference in their entirety. To elaborate, autologousserum samples were diluted 1:10, preadsorbed with lysates of E. colithat had been transfected with λ ZAP, further diluted to 1:200, and werethen incubated overnight at room temperature, with nitrocellulosemembranes containing phage plaques at a density of about 4-5000 pfus per130 mm plate.

[0012] The filters were washed, and then incubated with alkalinephosphatase conjugated, goat anti-human Fc γ secondary antibodies.Reactive phage plaques were visualized via incubation with5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium.

[0013] A total of 64 immunoreactive clones were identified. These weresubcloned, purified, and in vivo excised to pBK-CMV plasmid. Plasmidswere amplified, and DNA inserts were evaluated via EcoRI-Xba Irestriction mapping. Clones which represented different cDNA moleculesbased upon the mapping were sequenced using standard methodologies.

[0014] Analysis showed that 43 different genes were represented in thepositives. Ofthese, 29 were identical to, or highly homologous with,entries in GENBANK. The remaining 14 of these did not show homology withGENBANK sequences, but shared segments of identical sequences of ESTsfound in different issues.

[0015] Tables 1 and 2, which follow, summarize these results. TABLE 1Known genes identified by autologous SEREX screening of Mz19 cDNAlibrary Designation # of clones GenBank # Gene NY-MEL-1 1 [M94043]rab-related GTP-binding protein homolog NY-MEL-2 1 [AF109718]subtelomeric region chromosome 3 NY-MEL-3 1 [AF111423] XCAP-G homologNY-MEL-4 1 [D26156] trancriptional activator hSNF2b NY-MEL-5 3[NM002415] macrophage migration inhibiting factor NY-MEL-6 1 [AF047826]cadherin 7 NY-MEL-7 1 [AF091083] clone 628, chromosome X NY-MEL-8 2[NM000362] tissue inhibitor of metalloproteinase-3 NY-MEL-9 1 [NM004039]lipocortin II NY-MEL-10 1 [A8002374] KIAA 0376 NY-MEL-11 1 [NM002574]proliferation associated gene NY-MEL-12 2 [NM006141] dynein lightintermediate chain 2 NY-MEL-13 1 [NM001019] ribosomal protein S15aNY-MEL-14 1 [Z21852] HERV-K LTR NY-MEL-15 1 [X15183] HSP 90 NY-MEL-16 1[D80012] KIAA 0190 NY-MEL-17 1 [X07270] HSP 86 NY-MEL-18 1 [L07872]JK-recombination signal binding protein NY-MEL-19 1 [NM006227]phospholipid transfer protein NY-MEL-20 4 [NM004401] DNA fragmentationfactor, 45 kDa, alpha-subunit NY-MEL-21 1 [L22 154] ribosomal proteinL37a NY-MEL-22 1 [NM006395] ubiquitin activating enzyme El like proteinNY-MEL-23 1 [NM001064] transketolase NY-MEL-24 9 [U11687] 30S ribosomalprotein S1 homolog NY-MEL-25 1 [NM000994] ribosomal protein L32NY-MEL-26 1 [Z71183] cosmid N28H9 chromosome 22q11.2 NY-MEL-27 1[AF054183] GTP binding protein mRNA NY-MEL-28 1 [NM000358] TGF-betainduced gene product NY-MEL-29 1 [AV090384] SUMO-1

[0016] TABLE 2 Unknown genes identified by autologous SEREX screening ofMz19 cDNA library Designation # of clones EST sources/RT-PCR resultsNY-MEL-30 4 EST: total fetus, melanocytes; RT-PCR: ubiquitious NY-MEL-311 EST: brain, mammary gland, parathyroid tumor, colon, MS; RT-PCR:ubiquitious NY-MEL-32 1 EST: brain, pregnant uterus; RT-PCR: ubiquitiousNY-MEL-33 1 EST: tonsils (germinal center B cell enriched), melanocytes;RT-PCR: ubiquitious NY-MEL-34 1 EST: pregnant uterus, infant brain,fetal heart, CLL, eye, aorta, breast NY-MEL-35 1 EST: melanocytes,Wilm's tumor, tonsil, adrenal gland, fetal spleen NY-MEL-36 1 EST:prostate, fetal liver, colon tumor NY-MEL-37 1 EST: cerebellum, mouseskin, mouse mammary gland NY-MEL-38 1 EST: HeLa cells, mouse liverNY-MEL-39 1 EST: colon, ovarian cancer, testis, fibroblast NY-MEL-40 1EST: brain, retina, placenta, prostate cancer, pregnant uterus NY-MEL-411 EST: spleen, neuron, liver, Wilm's tumor NY-MEL-42 1 EST: kidney,melanocytes, breast, testis NY-MEL-43 1 EST: pregnant uterus,glioblastoma, pancreatic islets

EXAMPLE 2

[0017] The genes identified as NY-MEL-8 through NY-MEL-29 in table 1,supra, are known to be expressed over a wide range of human issues. Tenof the fourteen unknown genes exhibit ESTs isolated from several normaltissues, which suggests ubiquitous MRNA expression in adult tissues.Expression of the remaining seven known genes (MEL1-7) and the 4 unknowngenes (MEL30-33) was evaluated further. To do this, gene specificoligonucleotide primers were designed based upon the sequenceinformation. The primers were also designed to amplify segments 300-600base pairs in length, with primer melting temperatures of about 65-70°C. RT-PCR was then carried out using 30 amplification cycles, at anannealing temperature of 60° C. Products were analyzed by 1.5% gelelectrophoresis, and ethidium bromide visualization. Five representativenormal tissues were analyzed (brain, colon, kidney, testis, liver), withisolated clones serving as positive controls.

[0018] The results indicated that the four unknown genes were expresseduniversally, at similar levels. Of the 7 known genes, 5 of these (MEL-2,and MEL4-7) showed universal expression at similar levels. Two of thegenes, i.e., those identified as MEL-1 and MEL-3, showed differentiatedtissue expression. Specifically, MEL-1 (SEQ ID NO: 1 hereafter) gave astrong RT-PCR signal in melanoma line Mz19, weaker signals in testis andcancer, and was negative in brain, colon and liver. The gene representedby MEL-3 (SEQ ID NO: 2 hereafter) gave a strong RT-PCR signal in testisonly, with weak, albeit positive signals, in brain, colon, liver andkidney.

EXAMPLE 3

[0019] The mRNA expression of SEQ ID NO: 1 suggested further evaluationin a larger panel of normal tissues. This was carried out via Northernblot analyses. Commercially available poly A (2 μg/lane) multiple humantissue Northern blotting kits were used. Blotting was carried out usingrandomly primed, ³²p labelled probes, based upon the PCR productsdescribed supra. Hybridization took place for 60 minutes at 68° C.,followed by a high stringency wash (40 minutes, 2×SSC/0.05% SDS at roomtemperature, followed by 40 minutes in 0.1×SSC/0.1% SDS at 50° C.),followed by autoradiography. Northern blotting was also carried out ondifferent melanoma cell lines, using 10 μg/lane of total RNA.

[0020] No visible signal was found in any of the 17 normal tissuesamples tested. When expression in cells of melanocytic lineage wasexamined, a strong signal was found for an mRNA species of about 1.6kilobases in cultured melanocytes, and variable, weaker signals in fiveof seven melanoma cell lines tested. The predominant expressionsuggested than SEQ ID NO: 1 encodes a melanocyte differentiationantigen.

EXAMPLE 4

[0021] The results developed supra suggested than a simpler method fortyping for expression of SEQ ID NO: 1 in tumor samples was warranted.Hence, an RT-PCR assay was carried out on eight melanoma cell lines,using the protocol described supra. Seven of the cell lines werepositive for expression of SEQ ID NO: 1, using the standards supra.These results were compared to Northern blot analyses. Two of the lineswere positive for both Northern blotting and RT-PCR. Two culturedmelanocytes were also strongly positive in RT-PCR. Six breast cancerlines were negative by RT-PCR.

[0022] These results suggested that RT-PCR is more sensitive thanNorthern blotting for determining expression of SEQ ID NO: 1. This wasconfirmed when normal tissue RNA was examined via RT-PCR. SeveralNorthern blot negative tissues gave weak to moderate signals, includingtestis, kidney, uterus, prostate, and pancreatic tissue. Adrenal gland,which was not included in Northern blotting work, gave a positive RT-PCRsignal comparable to that for cultured melanocytes.

[0023] Following this work, a panel of tumor tissues was examined viaRT-PCR, also as described supra, of nineteen melanoma tumor specimensanalyzed, 17 were positive. Twelve gave a moderate to strong signal. Onelung cancer sample was positive, while 4 additional lung cancer, 4 coloncancer, 4 breast cancer and 4 squamous carcinomas were negative.

[0024] Analysis of SEQ ID NO: 1 shows a 1407 base pair molecule. Thefirst 47 5′ base pairs are not translated, as is also the case for thefinal 724 base pairs, which are followed by a polyA sequence 4nucleotides long. This provides an open reading frame of 636 base pairs,encoding a putative polypeptide of 211 amino acids, with a predictedmolecular mass of 23,714 kilodaltons.

[0025] Protein motif analysis shows five highly conserved GTP-bindingdomains, typical of ras superfamily members (ras, rab, rho, ran, arf). Acomparison with the GENBANK database confirmed homology to rat,rab-related GTP binding protein, with 81.5% identity at the nucleotidelevel, and 97% identity at the amino acid level.

[0026] With respect to human molecules, SEQ ID NO: 1 is closest inidentity to rab 32 (GENBANK NM006834), with 75% amino acid identity, and88% homology when standard conservative changes are made.

[0027] The protein encoded by SEQ ID NO: 1 (SEQ ID NO: 3 gives its aminoacid sequence), is unique in its carboxy terminus. Generally, rabproteins terminate in a “CC”, “CXC” or “CCXX” motif, which permitsposttranslational lipid modification, probably as geranylgeranylprenylation of the cysteine residues. This hydrophobic modification isfunctionally crucial for interaction with lipid membranes. The aminoacid sequence of SEQ ID NO: 3, however, terminated with CSGCAKS, whichmore resembles a ras protein than a rab protein. This suggests adifferent lipid modification pattern, probably farnesyl prenylation atcarboxy terminal cysteine, and palmitoylation at the upstream cysteine.See, e.g., Der, Meth. Enzymol 255: 46-60 (1995).

EXAMPLE 5

[0028] SEQ ID NO: 2, as reported supra, also showed differentialexpression. Analysis of this molecule shows a 3198 base pair cDNA, ofwhich 68 base pairs at the 5′ end, and 82 at the 3′ end areuntranslated. The untranslated 3′ end is followed by a 44 nucleotidepolyA sequence. The ORF extends for 3048 base pairs, encoding apolypeptide of 1015 amino acids with calculated molecular weight of114.3 kDa.

[0029] A homology search showed SEQ ID NO: 2 to be the human counterpartof X. laevis chromosome associated polypeptide group G (GenbankAccession No. AF111423). This molecule is a chromosome condensationprotein, which is part of the 13S condensin complex formed during earlystages of mitosis. SEQ ID NO: 2 shows 28% nucleotide identity with AF111423, and 59% amino acid identity.

[0030] When expression of SEQ ID NO: 2 was evaluated via RT-PCR andNorthern blotting, as described supra, the strongest Northern blottingsignal was found in testis, and a weak signal was found in thymus. Noother signals were found in normal tissues. Melanoma lines showedvariable signals of weak to moderate intensity.

[0031] When RT-PCR results were studied, all RNA of Northern blotpositive samples tested were positive, with highest expression intestis.

[0032] The foregoing examples describe the isolation of nucleic acidmolecules which encode a cancer associated antigen. “Associated” is usedherein because while it is clear that the relevant molecule wasexpressed by several types of cancer, other cancers, not screenedherein, may also express the antigen.

[0033] The invention relates to nucleic acid molecules which encode theantigens encoded by, e.g., SEQ ID NOS: 1 and 2, as well as the antigensencoded thereby, such as the proteins with the amino acid sequences ofSEQ ID NO: 3, and that encoded by the reading frame of SEQ ID NO: 2, asdiscussed supra. It is to be understood that all sequences which encodethe recited antigen are a part of the invention.

[0034] Also a part of the invention are those isolated molecules whichhybridize under stringent conditions to SEQ ID NO: 2, or those whichboth hybridize to SEQ ID NO: 1, under stringent conditions and encode aprotein which has, at its C terminus, the amino acid sequence CSGCAKS.“Stringent conditions”, as used herein, refers to conditions at least asstringent as those referred to in, e.g., U.S. Pat. No. 5,342,774, atexample 29, the disclosure of which is incorporated by reference.

[0035] Also a part of the invention are expression vectors whichincorporate the nucleic acid molecules ofthe invention, in operablelinkage (i.e., “operably linked”) to a promoter. Construction of suchvectors, such as viral (e.g., adenovirus or Vaccinia virus) orattenuated viral vectors is well within the skill of the art, as is thetransformation or transfection of cells, to produce eukaryotic celllines, or prokaryotic cell strains which encode the molecule ofinterest. Exemplary of the host cells which can be employed in thisfashion are COS cells, CHO cells, yeast cells, insect cells (e.g.,Spodoptera frugiperda), NIH 3T3 cells, and so forth. Prokaryotic cells,such as E. coli and other bacteria may also be used. Any of these cellscan also be transformed or transfected with further nucleic acidmolecules, such as those encoding cytokines, e.g., interleukins such asIL-2, 4, 6, or 12 or HLA or MHC molecules.

[0036] Also a part of the invention are the antigens described herein,both in original form and in any different post translational modifiedforms. The molecules are large enough to be antigenic without anyposttranslational modification, and hence are useful as immunogens, whencombined with an adjuvant (or without it), in both precursor andpost-translationally modified forms. Antibodies produced using theseantigens, both poly and monoclonal, are also a part of the invention aswell as hybridomas which make monoclonal antibodies to the antigens. Thewhole protein can be used therapeutically, or in portions, as discussedinfra. Also a part of the invention are antibodies against this antigen,be these polyclonal, monoclonal, reactive fragments, such as Fab,(F(ab)₂′ and other fragments, as well as chimeras, humanized antibodies,recombinantly produced antibodies, and so forth.

[0037] As is clear from the disclosure, one may use the proteins andnucleic acid molecules of the invention diagnostically. The SEREXmethodology discussed herein is premised on an immune response to apathology associated antigen. Hence, one may assay for the relevantpathology via, e.g., testing a body fluid sample of a subject, such asserum, for reactivity with the antigen per se. Reactivity would bedeemed indicative of possible presence of the pathology. So, too, couldone assay for the expression of any of the antigens via any of thestandard nucleic acid hybridization assays which are well known to theart, and need not be elaborated upon herein. One could assay forantibodies against the subject molecules, using standard immunoassays aswell.

[0038] Analysis of SEQ ID NOS: 1 and 2 will show that there are 5′ and3′ non-coding regions presented therein. The invention relates to thoseisolated nucleic acid molecules which contain at least the codingsegment, and which may contain any or all of the non-coding 5′ and 3′portions.

[0039] Also a part of the invention are portions of the relevant nucleicacid molecules which can be used, for example, as oligonucleotideprimers and/or probes, such as one or more of: cgaagagcag cataggaaagagttag gacactgtgt ttcacgttgg tc

[0040] (SEQ ID NOS: 4 and 5). These are the oligonucleotides used in theRT-PCR experiments for amplifying SEQ ID NO: 1, described supra.

[0041] As was discussed supra, study of other members of the “CT” familyreveals that these are also processed to peptides which provoke lysis bycytolytic T cells. There has been a great deal of work on motifs forvarious MHC or HLA molecules, which is applicable here. Hence, a furtheraspect of the invention is a therapeutic method, wherein one or morepeptides derived from the antigens of the invention which bind to an HLAmolecule on the surface of a patient's tumor cells are administered tothe patient, in an amount sufficient for the peptides to bind to theMHC/HLA molecules, and provoke lysis by T cells. Any combinationofpeptides may be used. These peptides, which may be used alone or incombination, as well as the entire protein or immunoreactive portionsthereof, may be administered to a subject in need thereof, using any ofthe standard types of administration, such as intravenous, intradermal,subcutaneous, oral, rectal, and transdermal administration. Standardpharmaceutical carriers, adjuvants, such as saponins, GM-CSF, andinterleukins and so forth may also be used. Further, these peptides andproteins may be formulated into vaccines with the listed material, asmay dendritic cells, or other cells which present relevant MHC/peptidecomplexes.

[0042] Similarly, the invention contemplates therapies wherein nucleicacid molecules which encode the proteins of the invention, one or moreor peptides which are derived from these proteins are incorporated intoa vector, such as a Vaccinia or adenovirus based vector, to render ittransfectable into eukaryotic cells, such as human cells. Similarly,nucleic acid molecules which encode one or more of the peptides may beincorporated into these vectors, which are then the major constituent ofnucleic acid bases therapies.

[0043] Any of these assays can also be used in progression/regressionstudies. One can monitor the course of abnormality involving expressionof these antigens simply by monitoring levels of the protein, itsexpression, antibodies against it and so forth using any or all of themethods set forth supra.

[0044] It should be clear that these methodologies may also be used totrack the efficacy of a therapeutic regime. Essentially, one can take abaseline value for a protein of interest using any of the assaysdiscussed supra, administer a given therapeutic agent, and then monitorlevels of the protein thereafter, observing changes in antigen levels asindicia of the efficacy of the regime.

[0045] As was indicated supra, the invention involves, inter alia, therecognition of an “integrated” immune response to the molecules oftheinvention. One ramification ofthis is the ability to monitor the courseof cancer therapy. In this method, which is a part of the invention, asubject in need of the therapy receives a vaccination of a typedescribed herein. Such a vaccination results, e.g., in a T cell responseagainst cells presenting HLA/peptide complexes on their cells. Theresponse also includes an antibody response, possibly a result of therelease of antibody provoking proteins via the lysis of cells by the Tcells. Hence, one can monitor the effect of a vaccine, by monitoring anantibody response. As is indicated, supra, an increase in antibody titermay be taken as an indicia of progress with a vaccine, and vice versa.Hence, a further aspect of the invention is a method for monitoringefficacy of a vaccine, following administration thereof, by determininglevels of antibodies in the subject which are specific for the vaccineitself, or a large molecule of which the vaccine is a part.

[0046] The identification of the subject proteins as being implicated inpathological conditions such as cancer also suggests a numberoftherapeutic approaches in addition to those discussed supra. Theexperiments set forth supra establish that antibodies are produced inresponse to expression of the protein. Hence, a further embodiment ofthe invention is the treatment of conditions which are characterized byaberrant or abnormal levels of one or more of the proteins, viaadministration of antibodies, such as humanized antibodies, antibodyfragments, and so forth. These may be tagged or labelled withappropriate cystostatic or cytotoxic reagents.

[0047] T cells may also be administered. It is to be noted that the Tcells may be elicited in vitro using immune responsive cells such asdendritic cells, lymphocytes, or any other immune responsive cells, andthen reperfused into the subject being treated.

[0048] Note that the generation of T cells and/or antibodies can also beaccomplished by administering cells, preferably treated to be renderednon-proliferative, which present relevant T cell or B cell epitopes forresponse, such as the epitopes discussed supra.

[0049] The therapeutic approaches may also include antisense therapies,wherein an antisense molecule, preferably from 10 to 100 nucleotides inlength, is administered to the subject either “neat” or in a carrier,such as a liposome, to facilitate incorporation into a cell, followed byinhibition of expression of the protein. Such antisense sequences mayalso be incorporated into appropriate vaccines, such as in viral vectors(e.g., Vaccinia), bacterial constructs, such as variants ofthe known BCGvaccine, and so forth.

[0050] Other features and applications ofthe invention will be clear tothe skilled artisan, and need not be set forth herein. The terms andexpression which have been employed are used as terms of description andnot of limitation, and there is no intention in the use of such termsand expression of excluding any equivalents of the features shown anddescribed or portions thereof, it being recognized that variousmodifications are possible within the scope of the invention.

1 5 1 1407 DNA Homo sapiens Unsure 1165 .. 1390 1 ggctgcgctt ccctggtcaggcacggcacg tctggccggc cgccaggatg caggccccgc 60 acaaggagca cctgtacaagttgctggtga ttggcgacct gggcgtgggg aagaccagta 120 tcatcaagcg ctacgtgcaccagaacttct cctcgcacta ccgggccaca atcggcgtgg 180 acttcgcgct caaggtgctccactgggacc cggagactgt ggtgcgcctg cagctctggg 240 atatcgcagg tcaagaaagatttggaaaca tgacgagggt ctattaccga gaagctatgg 300 gtgcatttat tgtcttcgatgtcaccaggc cagccacatt tgaagcagtg gcaaagtgga 360 aaaatgattt ggactccaagttaagtctcc ctaatggcaa accggtttca gtggttttgt 420 tggccaacaa atgtgaccaggggaaggatg tgctcatgaa caatggcctc aagatggacc 480 agttctgcaa ggagcacggtttcgtaggat ggtttgaaac atcagcaaag gaaaatataa 540 acattgatga agcctccagatgcctggtga aacacatact tgcaaatgag tgtgacctaa 600 tggagtctat tgagccggacgtcgtgaagc cccatctcac atcaaccaag gttgccagct 660 gctctggctg tgccaaatcctagtaggcac ctttgctggt gtctggtagg aatgacctca 720 ttgttccaca aattgtgcctctatttttac cattttgggt aaacgtcagg atagatatac 780 cacatgtggc aagccaaagatctatgcctc tgttttttca atgagagaga aatagcaaat 840 gttctttcta tgctttcctcaccatcatca cagtgtttac aaacttttga aaatatttag 900 tctgttacaa acttctgtcatgtagctgac caaaatcctg cagggccaca gtcggcactg 960 ttatttgctt cttttaatcagcaaaggcct caagtcttaa aataaaaggg gagaagaaca 1020 aactagctgt caagtcaaggactggctttc accttgccct ggtgtctttt tccagatttc 1080 aatatattct ctgatggcctgacaggccta ttaagtagat gtgatatttt cttccaagat 1140 gacctccatt ctcggcagacctaanaagtt gcctctgagt tagctctttg gaatcgngaa 1200 cacaggtgtg ctatattgnccttgtctaac tgncacttgn catggcctga atgttggctt 1260 aactgaatat tnnatgaaaagacatgcctt catatgtgcc tttttggtaa ctttctttga 1320 ctnaacctng gggctcctttttcatgcttt acatgtaaaa tatatatttt ttttttgcag 1380 gggaccattn aacctttaaggataaaa 1407 2 3242 DNA Homo sapiens Unsure 3037 .. 3198 2 ctgggttggcgggctggcag gctgtagccg agcgcgggca ggactcgtcc cggcagggtt 60 ccagagccatgggagcggaa aggaggctgc tgtcgattaa ggaggccttt cggctggcgc 120 agcagccgcaccagaaccag gcgaagctgg tggtggcgct gagccgcacc taccgcacga 180 tggatgataagacagttttt catgaggagt tcattcatta ccttaaatat gttatggtgg 240 tctataaacgtgaaccagct gtggagaggg taatagaatt tgcagcaaag tttgttacct 300 catttcaccaatcagatatg gaagatgatg aggaagagga agatggtggc cttttaaatt 360 atttgtttacttttctctta aagtctcatg aagcaaacag caatgcagtg agatttagag 420 tgtgcctgctcataaacaag cttttgggaa gtatgccaga aaatgctcag attgatgatg 480 atgtgtttgataaaattaat aaagccatgc ttattagatt gaaagataag attccaaatg 540 tgagaatacaggcagttctg gcgctttcac gacttcagga tcccaaggat gatgaatgcc 600 cagtggttaatgcatatgct actttgattg aaaatgattc aaatccagaa gttagacggg 660 cagtgttatcatgtattgca ccatcagcaa agactttgcc aaaaattgta gggcgcacca 720 aggatgtgaaagaggctgtc agaaagctgg cttatcaggt tttagctgaa aaggttcata 780 tgagagctatgtccattgct cagagagtaa tgctccttca acaaggtctt aatgacagat 840 cagatgctgtgaaacaagct atgcagaagc atcttcttca aggctggtta cgggtctctg 900 aaggaaatatcttagagttg ctccatcggt tggatgtaga aaattcttct gaagtggcag 960 tctctgttctcaatgccttg ttttcaataa ctcctctcag tgaactggtg ggactctgta 1020 aaaacaatgatggcaggaaa ttgattccag tggaaacatt aactcctgaa attgctttgt 1080 attggtgtgccctttgtgaa tatttgaaat caaaaggaga tgaaggtgaa gaatttttag 1140 agcagattttgccagagcct gtagtatatg cagactattt attgagttac atccagagca 1200 ttccagttgttaatgaagaa cacagaggtg atttttccta tattggaaat ttgatgacaa 1260 aagaattcataggtcaacaa ttgattctaa ttattaagtc tttggatacc agtgaagaag 1320 gaggaagaaaaaaactgctg gctgttttac aggagattct tattttaccc acaatcccaa 1380 tatccctggtttcttttctt gttgaaagac tactccacat cattatagat gataataaga 1440 gaacacaaattgttacagaa attatctcag agattcgggc gcccattgtt actgttggtg 1500 ttaataacgatccagctgat gtaagaaaga aagaactcaa gatggctgaa ataaaagtta 1560 agcttatcgaagccaaagaa gctttggaaa attgcattac cttacaggat tttaatcggg 1620 catcagaattaaaagaagaa ataaaagcat tagaagatgc cagaataaac cttttgaaag 1680 agacagagcaacttgaaatt aaagaagtcc acatagagaa gaatgatgct gaaacattgc 1740 agaaatgtcttattttatgc tatgaactgt tgaagcagat gtccatttca acaggcttaa 1800 gtgcaaccatcaatggaatc atcgaatctt tgattcttcc tggaataata agtattcatc 1860 ctgttgtaagaaacctggct gttttatgct tgggatgctg tggactacag aatcaggatt 1920 ttgcaaggaaacacttcgta ttactattgc aggttttgca aattgatgat gtcacaataa 1980 aaataagtgctttaaaggca atctttgacc aactgatgac gttcgggatt gaaccattta 2040 aaactaaaaaaatcaaaaca cttcattgtg aaggtacaga aataaacagt gatgatgagc 2100 aagaatcaaaagaagttgaa gagactgcta cagctaagaa tgttctgaaa ctcctttctg 2160 atttcttagatagtgaggta tctgaactta ggactggagc tgcagaagga ctagccaagc 2220 tgatgttctctgggcttttg gtcagcagca ggattctttc tcgtcttatt ttgttatggt 2280 acaatcctgtgactgaagag gatgttcaac ttcgacattg cctaggcgtg ttcttccccg 2340 tgtttgcttatgcaagcagg actaatcagg aatgctttga agaagctttt cttccaaccc 2400 tgcaaacactggccaatgcc cctgcatctt ctcctttagc tgaaattgat atcacaaatg 2460 ttgctgagttacttgtagat ttgacaagac caagtggatt aaatcctcag gccaagactt 2520 cccaagattatcaggcctta acagtacatg acaatttggc tatgaaaatt tgcaatgaga 2580 tcttaacaagtccgtgctcg ccagaaattc gagtctatac aaaagccttg agttctttag 2640 aactcagtagccatcttgca aaagatcttc tggttctatt gaatgagatt ctggagcaag 2700 taaaagataggacatgtctg agagctttgg agaaaatcaa gattcagtta gaaaaaggaa 2760 ataaagaatttggtgaccaa gctgaagcag cacaggatgc caccttgact acaactactt 2820 tccaaaatgaagatgaaaag aataaagaag tatatatgac tccactcagg ggtgtaaaag 2880 caacccaagcatcaaagtct actcagctaa agactaacag aggacagaga aaagtgacag 2940 tttcagctaggacgaacagg aggtgtcaga ctgctgaagc cgactctgaa agtgatcatg 3000 aagttccagaaccagaatca gaaatgaaga tgagactncc aagacgagcc aaaaccgcag 3060 cnctanaaaaaagtaaactt anccttgccc aatttttcaa tgaagattta agttaggaaa 3120 nacgatggaggnggaatcct ttaagattat gtccagttat ttgctttaat aaanaanaag 3180 ttacccttgtcaaaatcnaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3240 aa 3242 3211 PRT Homo sapiens 3 Met Gln Ala Pro His Lys Glu His Leu Tyr Lys LeuLeu Val Ile Gly 1 5 10 15 Asp Leu Gly Val Gly Lys Thr Ser Ile Ile LysArg Tyr Val His Gln 20 25 30 Asn Phe Ser Ser His Tyr Arg Ala Thr Ile GlyVal Asp Phe Ala Leu 35 40 45 Lys Val Leu His Trp Asp Pro Glu Thr Val ValArg Leu Gln Leu Trp 50 55 60 Asp Ile Ala Gly Gln Glu Arg Phe Gly Asn MetThr Arg Val Tyr Tyr 65 70 75 80 Arg Glu Ala Met Gly Ala Phe Ile Val PheAsp Val Thr Arg Pro Ala 85 90 95 Thr Phe Glu Ala Val Ala Lys Trp Lys AsnAsp Leu Asp Ser Lys Leu 100 105 110 Ser Leu Pro Asn Gly Lys Pro Val SerVal Val Leu Leu Ala Asn Lys 115 120 125 Cys Asp Gln Gly Lys Asp Val LeuMet Asn Asn Gly Leu Lys Met Asp 130 135 140 Gln Phe Cys Lys Glu His GlyPhe Val Gly Trp Phe Glu Thr Ser Ala 145 150 155 160 Lys Glu Asn Ile AsnIle Asp Glu Ala Ser Arg Cys Leu Val Lys His 165 170 175 Ile Leu Ala AsnGlu Cys Asp Leu Met Glu Ser Ile Glu Pro Asp Val 180 185 190 Val Lys ProHis Leu Thr Ser Thr Lys Val Ala Ser Cys Ser Gly Cys 195 200 205 Ala LysSer 210 4 26 DNA Homo sapiens 4 cgaagagcag cataggaaag agttag 26 5 22 DNAHomo sapiens 5 gacactgtgt ttcacgttgg tc 22

We claim:
 1. An isolated nucleic acid molecule which encodes a cancerassociated antigen, the C terminal amino acid sequence of which isCSGCAKS, the complementary sequence of which hybridizes to SEQ ID NO: 1under stringent conditions.
 2. The isolated nucleic acid molecule ofclaim 1, comprising the nucleotide sequence of SEQ ID NO:
 1. 3. Theisolated nucleic acid molecule of claim 1, comprising the nucleotidesequence defined by nucleotides 48-683 of SEQ ID NO:
 1. 4. The isolatednucleic acid molecule of claim 1, comprising a nucleotide sequence whichencodes a protein, the amino acid sequence of which is set forth at SEQID NO:
 3. 5. An isolated nucleic acid molecule which encodes a cancerassociated antigen, the complementary sequence of which hybridizes toSEQ ID NO:
 2. 6. The isolated nucleic acid molecule of claim 5,comprising the nucleotide sequence of SEQ ID NO:
 2. 7. The isolatednucleic acid of claim 5, comprising nucleotides 69-3116 of SEQ ID NO: 2.8. The isolated nucleic acid molecule of claim 5, comprising anucleotides sequence that encodes the protein encoded by nucleotides69-3116 of SEQ ID NO:
 2. 9. Expression vector comprising the isolatednucleic acid molecule of claim 1, operably linked to a promoter. 10.Expression vector comprising the isolated nucleic acid molecule of claim5, operably linked to a promoter.
 11. Eukaryotic cell line orprokaryotic cell strain, transformed or transfected with the expressionvector of claim 9 or
 10. 12. Isolated cancer associated antigencomprising all or part of the amino acid sequence encoded by SEQ ID NO:1 or
 2. 13. The isolated cancer associated antigen of claim 12,comprising the amino acid sequence of SEQ ID NO: 3
 14. Eukaryotic cellline or prokaryotic cell strain, transformed or transfected with theisolated nucleic acid molecule of claim 1 or
 5. 15. The eukaryotic cellline or prokaryotic cell strain of claim 14, wherein said cell line isalso transfected with a nucleic acid molecule coding for a cytokine. 16.The eukaryotic cell line or prokaryotic cell strain of claim 15, whereinsaid cell line is further transfected by a nucleic acid molecule codingfor an MHC molecule.
 17. The eukaryotic cell line orprokaryotic cellstrain of claim 15, wherein said cytokine is an interleukin.
 18. Theeukaryotic cell line or prokaryotic cell strain of claim 17, whereinsaid interleukin is IL-2, IL-4 or IL-12.
 19. The eukaryotic cell line orprokaryoiic cell strain of claim 14, wherein said cell line has beenrendered non-proliferative.
 20. The eukaryotic cell line of claim 14,wherein said cell line is a fibroblast cell line.
 21. Expression vectorcomprising a mutated or attenuated virus and the isolated nucleic acidmolecule of claim 1 or
 5. 22. The expression vector of claim 21, whereinsaid virus is adenovirus or vaccinia virus.
 23. The expression vector ofclaim 22, wherein said virus is vaccinia virus.
 24. The expressionvector of claim 22, wherein said virus is adenovirus.
 25. Expressionsystem useful in transfecting a cell, comprising (i) a first vectorcontaining a nucleic acid molecule which codes for the isolated cancerassociated antigen of claim 12 and (ii) a second vector selected fromthe group consisting of (a) a vector containing a nucleic acid moleculewhich codes for an MHC or HLA molecule which presents an antigen derivedfrom said cancer associated antigen and (b) a vector containing anucleic acid molecule which codes for an interleukin.
 26. Immunogeniccomposition comprising the isolated cancer antigen of claim 12, and apharmaceutically acceptable adjuvant.
 27. The immunogenic composition ofclaim 26, wherein said adjuvant is a cytokine, a saponin, or GM-CSF. 28.Immunogenic composition comprising at least one peptide consisting of anamino acid sequence of from 8 to 12 amino acids concatenated to eachother in the isolated cancer associated antigen of claim 12, and apharmaceutically acceptable adjuvant.
 29. The immunogenic composition ofclaim 28, wherein said adjuvant is a saponin, a cytokine, or GM-CSF. 30.The immunogenic composition of claim 27, wherein said compositioncomprises a plurality of peptides which complex with a specific MHCmolecule.
 31. Immunogenic composition which comprises at least oneexpression vector which encodes a peptide derived from the amino acidsequence encoded by SEQ ID NO: 1 or
 2. 32. The immunogenic compositionof claim 31, wherein said at least one expression vector codes for aplurality of peptides.
 33. Vaccine useful in treating a subjectafflicted with a cancerous condition comprising the isolated eukaryoticcell line of claim 14 and a pharmacologically acceptable adjuvant. 34.The vaccine of claim 33, wherein said eukaryotic cell line has beenrendered non-proliferative.
 35. The vaccine of claim 34, wherein saideukaryotic cell line is a human cell line.
 36. A composition of matteruseful in treating a cancerous condition comprising a non-proliferativecell line having expressed on its surface a peptide derived from theamino acid sequence encoded by SEQ ID NO: 1 or
 2. 37. The composition ofmatter of claim 36, wherein said cell line is a human cell line.
 38. Acomposition of matter useful in treating a cancerous condition,comprising (i) a peptide derived from the amino acid sequence encoded bySEQ ID NO: 1 or 2, (ii) an MHC or HLA molecule, and (iii) apharmaceutically acceptable carrier.
 39. Isolated antibody which isspecific for the cancer antigen of claim
 12. 40. The isolated antibodyof claim 39, wherein said antibody is a monoclonal antibody.
 41. Methodfor screening for cancer in a sample, comprising contacting said samplewith a nucleic acid molecule which hybridizes to all or part of themolecule encoded by SEQ ID NO: 1 or 2 and determining hybridization asan indication of cancer cells in said sample.
 42. A method for screeningfor cancer in a sample, comprising contacting said sample with theisolated antibody of claim 39, and determining binding of said antibodyto a target as an indicator of cancer.
 43. Method for diagnosing acancerous condition in a subject, comprising contacting an immunereactive cell containing sample of said subject to a cell linetransfected with the isolated nucleic acid molecule of claim 1 or 5, anddetermining interaction of said transfected cell line with saidimmunoreactive cell, said interaction being indicative of said cancercondition.
 44. A method for determining regression, progression of onsetof a cancerous condition comprising monitoring a sample from a patientwith said cancerous condition for a parameter selected from the groupconsisting of (i) a protein encoded by SEQ ID NO: 1 or 2, (ii) a peptidederived from said protein, (iii) cytolytic T cells specific for saidpeptide and an MHC molecule with which it non-covalently complexes, and(iv) antibodies specific for said protein, wherein amount of saidparameter is indicative of progression or regression or onset of saidcancerous condition.
 45. The method of claim 44, wherein said sample isa body fluid or exudate.
 46. The method of claim 44, wherein said sampleis a tissue.
 47. The method of claim 44, comprising contacting saidsample with an antibody which specifically binds with said protein orpeptide.
 48. The method of claim 47, wherein said antibody is labelledwith a radioactive label or an enzyme.
 49. The method of claim 47,wherein said antibody is a monoclonal antibody.
 50. The method of claim44, comprising amplifying RNA which codes for said protein.
 51. Themethod of claim 50, wherein said amplifying comprises carrying outpolymerase chain reaction.
 52. The method of claim 44, comprisingcontacting said sample with a nucleic acid molecule which specificallyhybridizes to a nucleic acid molecule which codes for or expresses saidprotein.
 53. The method of claim 52, wherein said nucleic acid moleculecomprises SEQ ID NO: 4 or
 5. 54. The method of claim 44, comprisingassaying said sample for shed protein.
 55. The method of claim 44,comprising assaying said sample for antibodies specific for saidprotein, by contacting said sample with protein.
 56. Method fordiagnosing a cancerous condition comprising assaying a sample taken froma subject for an immunoreactive cell specific for a peptide derived froma protein encoded by SEQ ID NO: 1 or 2, complexed to an MHC molecule,presence of said immunoreactive cell being indicative of said cancerouscondition.
 57. Composition comprising at least one peptide consisting ofan amino acid sequence of from 8 to 25 amino acids concatenated to eachother in the isolated cancer associated antigen of claim 12, and apharmaceutically acceptable adjuvant.
 58. The composition of claim 57,wherein said adjuvant is a saponin, a cytokine, or GM-CSF.
 59. Thecomposition of claim 57, comprising a plurality of MHC binding peptides.60. Composition comprising an expression vector which encodes at leastone peptide consisting of an amino acid sequence of from 8 to 25 aminoacids concatenated to each other in the isolated cancer associatedantigen of claim 12, and pharmaceutically acceptable adjuvant.
 61. Thecomposition of claim 60, wherein said expression vector encodes aplurality of peptides.
 62. A method for screening for possible presenceof a pathological condition, comprising assaying a sample from a patientbelieved to have a pathological condition for antibodies specific to atleast one of the cancer associated antigens encoded by SEQ ID NO: 1 or2, presence of said antibodies being indicative of possible presence ofsaid pathological condition.
 63. The method of claim 62, wherein saidpathological condition is cancer.
 64. The method of claim 62, whereinsaid cancer is melanoma.
 65. The method of claim 64, further comprisingcontacting said sample to purified cancer associated antigen encoded bySEQ ID NO: 1 or
 2. 66. A method for screening for possible presence of apathological condition in a subject, comprising assaying a sample takenfrom said subject for expression of a nucleic acid molecule, thenucleotide sequence of which comprises SEQ ID NO: 1 or 2, expression ofsaid nucleic acid molecule being indicative of possible presence of saidpathological condition.
 67. The method of claim 66, wherein saidpathological condition is cancer.
 68. The method of claim 66, comprisingdetermining expression via polymerase chain reaction.
 69. The method ofclaim 66, comprising determining expression by contacting said samplewith at least one of SEQ ID NO: 4 or
 5. 70. A method for determiningregression, progression of onset of a cancerous condition comprisingmonitoring a sample from a patient with said cancerous condition for aparameter selected from the group consisting of (i) a cancer associatedantigen encoded by SEQ ID NO: 1 or 2, (ii) a peptide derived from saidcancer associated antigen, (iii) cytolytic T cells specific for saidpeptide and an MHC molecule with which it non-covalently complexes, and(iv) antibodies specific for said cancer associated antigen, whereinamount of said parameter is indicative of progression or regression oronset of said cancerous condition.
 71. The method of claim 70, whereinsaid sample is a body fluid or exudate.
 72. The method of claim 70,wherein said sample is a tissue.
 73. The method of claim 70, comprisingcontacting said sample with an antibody which specifically binds withsaid protein or peptide.
 74. The method of claim 73, wherein saidantibody is labelled with a radioactive label or an enzyme.
 75. Themethod of claim 73, wherein said antibody is a monoclonal antibody. 76.The method of claim 70, comprising amplifying RNA which codes for saidprotein.
 77. The method of claim 76, wherein said amplifying comprisescarrying out polymerase chain reaction.
 78. The method of claim 70,comprising contacting said sample with a nucleic acid molecule whichspecifically hybridizes to a nucleic acid molecule which codes for orexpresses said protein.
 79. The method of claim 70, comprising assayingsaid sample for shed cancer associated antigen.
 80. The method of claim70, comprising assaying said sample for antibodies specific for saidcancer associated antigen, by contacting said sample with said cancerassociated antigen.
 81. Method for screening for a cancerous conditioncomprising assaying a sample taken from a subject for an immunoreactivecell specific for a peptide derived from a cancer associated antigenencoded by SEQ ID NO: 1 or 2, complexed to an MHC molecule, presence ofsaid immunoreactive cell being indicative of said cancerous condition.